Peer Reviewed: Antimicrobial Testing

Antimicrobial Effectiveness Testing Validation Strategies

Crystal Booth

ABSTRACT use or storage. Antimicrobial agents may use of the product (3). The test is designed
The antimicrobial effectiveness test be harmful to patients. If possible, it is im- to provide a laboratory test that gauges the
(AET) is used to assess preservative efficacy portant to demonstrate that the agents are level of biological activity possessed by the
of products in multi-dose containers. The effective in the final package and that they preservative system of a product (3). The
preservative efficacy of products can ei- are safe for human use. Products which test is used during product development
ther be due to added preservatives, or the have inherent antimicrobial properties to determine the effectiveness of the prod-
inherent properties of the product without must also be analyzed for antimicrobial uct and during stability to demonstrate the
the addition of preservatives (1). The test effectiveness. Antimicrobial effectiveness preservative system is stable over time. The
is not intended to challenge the ability of must be demonstrated for all products mul- test is not designed to be a quality control
product in multi-dose containers to with- tidose containers. (1) release test. The test must not be used as
stand in-use contamination (2). Obtaining a substitute for good manufacturing prac-
successful validation of the assay can be The article presented here provides in- tices.
complex depending on the product. Meth- sight to the test and strategies for obtaining
od development trials can be streamlined a successful validation of the test. The test involves inoculating a measured
to produce an adequate method by work- amount of product with known amounts of
ing closely with development scientists, APPLICATION OF THE ANTIMI- microorganisms. Whenever possible, the
chemists, setting appropriate specifications, original containers are utilized for the as-
CROBIAL EFFECTIVENESS TEST
and performing research. Key factors in say. The containers are protected from light
The antimicrobial effectiveness test is
developing a proper method include some and incubated at ambient temperature for
used to evaluate the effectiveness of preser-
experimentation as well as knowledge of 28 days. The death rate is measured over a
vative systems in multidose dosage forms.
the test material properties. This article 28 day period and compared to the accep-
Products in multidose forms that do not
discusses strategies to assist in obtaining tance criteria outlined in the compendial
contain added preservatives must still com-
successful validation of the assay. guidance documents.
ply with the test to demonstrate that the
inherent antimicrobial properties of the
INTRODUCTION Certain microorganisms can adversely
product are effective. AET is a tripartite
The antimicrobial effectiveness test (also impact (reduce or inactivate) the activity
compendial test performed during devel-
known as the preservative effectiveness of certain products (10). It is important
opment and stability testing of parenteral
test) first appeared as a USP General Chap- to understand the microbial load of the
products intended as a multidose product
ter in the 18th revision, official September non-sterile finished products in order to
(7).
1, 1970 (3). International harmonization determine if the product will react the way
has not been completed to date. The United it was intended. Likewise, it is important
The antimicrobial effectiveness test was
States Pharmacopeia (USP), Japanese Phar- to understand the effectiveness of the pre-
originally designed to evaluate the perfor-
macopeia (JP), and European Pharmaco- servative system to ensure the product will
mance of antimicrobials added to inhibit
peia (EP) all describe the assay in a similar remain active as intended overtime without
the growth of microorganisms that might
fashion, but the acceptance criterion varies garnering microorganisms which can cause
be introduced in the product during or sub-
amongst the different compendial chapters. human infection.
sequent to the manufacturing process (8).
Since 1970, the purpose of the test has been
Antimicrobial preservatives are sub- The guidance documents listed in Table
refined to focus on activity of the preserva-
stances that are added to products to limit 1 govern the Antimicrobial Effectiveness
tive systems as a protection against inad-
microbial contamination during normal Test. This may not be an all-inclusive list.
vertent contamination during storage and

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Crystal Booth

Table 1: Governing Documents for the Amtimicrobial Effectivess Test

REFERENCE DOCUMENT TITLE

USP <51> Antimicrobial Effectiveness Test (AET)
EP 5.1.3. Efficacy of Antimicrobial Preservation
JP 19 Preservatives-Effectiveness Tests (PET)
ICH Guidance Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug
Substances and New Drug Products: Chemical Substances

Respective USP <51>, EP 5.1.3, and JP 19 tests are not harmonized. The assays them-
selves are similar, but the acceptance criteria differ in each compendium. Table 2 is a
comparison table of the compendial chapters. The compromise column of Table 2 is a
stepwise comparison with recommendations to develop one method that can be utilized
for compliance with all three compendia.

Table 2: Antimicrobial Effectivess Testing Comparison

REQUIREMENT USP <51> JP 19 EP 5.1.3 COMPROMISE

REASON FOR TEST Antimicrobial Effectiveness, To assess microbiologically The antimicrobial activity of This is just verbiage differences
whether inherent in the the preservative efficacy, either the preparation in its final between the compendiums and
product or produced because due to the action of product container is investigated over do not affect the test method.
of a preservative, must be components themselves or the period of validity to ensure
demonstrated for all injections any added preservative(s), for that such activity has not been
packaged in multiple-dose multi-dose containers. Tests impaired by storage. Such in-
containers or for other prod- are commonly used to verify vestigations may be carried on
ucts containing antimicrobial that products maintain their samples removed from the final
preservatives. Antimicrobial preservative effectiveness container immediately prior to
Effectiveness must be demon- at the design phase of the testing. During development
strated for multiple-dose formulation or in the case of of a pharmaceutical prepara-
topical and oral dosage forms periodic monitoring. Tests are tion, it shall be demonstrated
and for other dosage forms, not performed for lot release; that the antimicrobial activity
ophthalmic, otic, nasal, irriga- the efficacy of the preservative of the preparation provides
tion, and dialysis fluids. present in the product pack- adequate protection from
aged in the final containers adverse effects that may arise
should be verified throughout from microbial contamination
the entire dating period. or proliferation during storage
and use of the preparation.
The test is not intended for
routine control purposes.

CATEGORIES OF PRODUCT. 1) Injections, other parenter- 1) Products are those made Parenteral and Ophthalmic This section describes the
CRITERIA OF ANTIMI- als including emulsions, otic with aqueous bases or vehicles. Preparations different category of products.
CROBIAL EFFECTIVENESS products, sterile nasal products, (Oil-in water Emulsions) 1A] It does not affect the test meth-
ARE A FUNCTION OF THE and ophthalmic products made Injections and other sterile par- od, but instead of calling the
ROUTE OF ADMINISTRA- with aqueous bases or vehicles. enterals. 1B] Nonsterile Paren- products category 1, etc. The
TION terals. 1C] Oral products made products should be described
with aqueous bases (including by what they are, topical non-
syrup products to be dissolved aqueous, etc.
or suspended before use).

2) Topically used products 2) Products made with non- Topical preparations

made with aqueous bases or aqueous bases or vehicles. (Wa-
vehicles, nonsterile nasal prod- ter-in-Oil Emulsions). All the
ucts, and emulsions, including dosage forms listed in Category
those applied to mucous 1 but made with nonaqueous
membranes. bases or vehicles.

3) Oral products other than NA Oral preparations

antacids, made with aqueous
bases or vehicles.

Journal of GXP Compliance Volume 18 Number 3

Peer Reviewed: Antimicrobial Testing
CATEGORIES OF PRODUCT 4) Antacids made with an NA NA This section describes the
aqueous base. different category of products.
It does not affect the test meth-
od, but instead of calling the
products category 1, etc. The
products should be described
by what they are, topical non-
aqueous, etc.

TEST ORGANISMS Candida albicans ATCC 10231, Candida albicans ATCC 10231, Candida albicans ATCC 10231, Candida albicans ATCC 10231,
Aspergillus brasiliensis ATCC Aspergillus brasiliensis ATCC Aspergillus brasiliensis ATCC Aspergillus brasiliensis ATCC
16404, Escherichia coli ATCC 16404, Escherichia coli ATCC 16404, Pseudomonas aerugino- 16404, Escherichia coli ATCC
8739, Pseudomonas aeruginosa 8739, Pseudomonas aeruginosa sa ATCC 9027, Staphylococcus 8739, Pseudomonas aeruginosa
ATCC 9027, Staphylococcus ATCC 9027, Staphylococcus aureus ATCC 6538 ATCC 9027, Staphylococcus
aureus ATCC 6538 aureus ATCC 6538 aureus ATCC 6538

Must not be more than 5 pas- Must not be more than 5 pas- It is recommended to keep Must not be more than 5 pas-
sages from the original ATCC sages from the original culture number of transfers to a sages from the original culture
culture minimum.

NA In addition to ATCC strains, it Single strains are used and In addition to ATCC strains, it
is recommended to use strains the designated microorgan- is recommended to use strains
that might contaminate the isms can be substituted with that might contaminate the
product and grow on or in it, environmental isolates. It is product and grow on or in it,
depending on its character- recommended that E. coli depending on its character-
istics. ATCC 8739 be used for oral istics.
preparations and Zygosaccha-
raomyces rouxii NCY 381 for
oral preparations containing a
high concentration of sugar.

MEDIA Must be growth promoted NA NA Must be growth promoted

using the test microorganisms. using the test microorganisms.

PREPARATION OF Inoculate surface of solid agar Inoculate surface of solid agar Inoculate the surface of agar Inoculate surface of solid agar
INOCULUM with each specified microor- with each specified microor- medium B for bacteria or agar with each specified microor-
ganism. Either broth or solid ganism. Either broth or solid medium C without the addi- ganism. Either broth or solid
media may be used for the media may be used for the tion of antibiotics for fungi media may be used for the
harvest. harvest. harvest.

E. coli 32.5 + 2.5ºC for 18-24 E. coli 32.5 + 2.5ºC for 18-24 NA E. coli 32.5 + 2.5ºC for 18-24
hours/ TSA hours/ TSA hours/ TSA

P. aerugionosa 32.5 + 2.5ºC for P. aerugionosa 32.5 + 2.5ºC for P. aerugionosa 32.5 + 2.5ºC for P. aerugionosa 32.5 + 2.5ºC for
18-24 hours/ TSA 18-24 hours/ TSA 18-24 hours/ agar medium B 18-24 hours/ TSA

S. aureus 32.5 + 2.5ºC for 18-24 S. aureus 32.5 + 2.5ºC for 18-24 S. aureus 32.5 + 2.5ºC for 18-24 S. aureus 32.5 + 2.5ºC for 18-24
hours/ TSA hours/ TSA hours/ agar medium B hours/ TSA

C. albicans 22.5 + 2.5ºC for C. albicans 22.5 + 2.5ºC for C. albicans 22.5 + 2.5ºC for C. albicans 22.5 + 2.5ºC for
44-52 hours/ SDA 40-48 hours/ SDA 48 hours/ agar medium C 40-48 hours/ SDA

A. brasiliensis 22.5 + 2.5ºC for A. brasiliensis 22.5 + 2.5ºC for 1 A. brasiliensis 22.5 + 2.5ºC for 1 A. brasiliensis 22.5 + 2.5ºC for
6-10 days/ SDA week or until good sporulation week or until good sporulation 6-10 days/ SDA
is achieved/ SDA is achieved/ agar medium C

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HARVESTING MICROBES Harvest bacterial and yeast Harvest cultured cells asepti- Harvest bacterial and yeast Harvest bacterial and yeast
with sterile saline TS, wash sur- cally using a platinum loop, etc. with sterile saline TS, wash sur- with sterile saline TS, wash sur-
face with and collect in a vessel Suspend in sterile physiological face with and collect in a vessel face with and collect in a vessel
to obtain ~1X 108 CFU/Ml saline or in 0.1% peptone water to obtain ~1X 108 CFU/mL to obtain ~1X 108 CFU/mL
and adjust to ~1X 108 CFU/mL

Harvest mold with sterile Harvest mold with sterile Harvest mold with sterile Harvest mold with sterile
saline TS containing 0.05% physiological saline or 1% saline TS containing 0.05% saline TS containing 0.05%
of polysorbate 80 and collect peptone water containing 0.05 of polysorbate 80 and collect of polysorbate 80 and collect
in a vessel to obtain ~1X 108 w/v% of polysorbate 80 and in a vessel to obtain ~1X 108 in a vessel to obtain ~1X 108
CFU/mL adjust to a spore count of ~1X CFU/mL CFU/mL
108 CFU/mL

May also grow microorganisms May also grow microorganisms NA May also grow microorganisms
in Broth in Broth in Broth

Confirm the estimate of the Titrate the viable cell count Confirm the estimate of the Titrate the viable cell count
suspensions. of the inoculum immediately suspensions. Use the plate of the inoculum immediately
before use, and then calculate count or membrane filtration before use, and then calculate
the theoretical viable cell count method. the theoretical viable cell count
per mL (g) of the product just per mL (g) of the product just
after inoculation. after inoculation.

Use bacteria and yeast within Use bacteria and yeast within The suspension shall be used Use the suspensions imme-
24 hours. Refrigerate if not 24 hours. Refrigerate if not immediately. diately.
used within 2 hours. Mold may used within 2 hours.
be refrigerated for 7 days.

NA If using isolates, other media NA If using isolates, other media

may be used depending on the may be used depending on the
isolate. isolate.

PROCEDURE Conduct test in 5 original Inject each of the cell suspen- Inoculate a series of containers Conduct test in 5 original
containers if sufficient volume sions aseptically into 5 con- of the product to be examined, containers if sufficient volume
of product is available in each tainers containing the product each with a suspension of one of product is available in each
container and the container can and mix uniformly if sufficient of the test microorganisms. container and the container
be entered aseptically. volume of product is available. can be entered aseptically.
For nonsterile products, Also incubate product controls
incubate a product control and without microbes.
determine the viable counts.

Inoculate each container with The volume used must not ex- Inoculate each container with Inoculate each container with
one inoculum suspension and ceed 1/100 of the volume of the one inoculum suspension and one inoculum suspension and
mix. The volume of inoculum product. Final concentration mix. The volume of inoculum mix. The volume of inoculum
is to be between 0.5% and 1.0% is between 1X 105 and 1X 106 does not exceed 1.0% of the is to be between 0.5% and
of the volume of product. Final CFU/mL of product. volume of product. Final 1.0% of the volume of product.
concentration is between 1X concentration is between 1X Final concentration is between
105 and 1X 106 CFU/mL of 105 and 1X 106 CFU/mL of 1X 105 and 1X 106 CFU/mL
product for Categories 1-3. product. of product for all products
Category 4 between 1X 103 and but Antacids made with an
1X 104 CFU/mL of product. aqueous base. For Antacids
made with an aqueous base
final concentration is between
1X 103 and 1X 104 CFU/mL of
product.

Incubate the inoculated Incubate the inoculated Incubate the inoculated Incubate the inoculated con-
containers at 22.5 + 2.5ºC and containers at 22.5 + 2.5ºC and containers at 22.5 + 2.5ºC (pro- tainers at 22.5 + 2.5ºC (protect-
sample at the appropriate inter- sample at the appropriate inter- tected from light) and sample ed from light) and sample at
vals. Category 1: 7, 14, 28 days/ vals protected from light. Test at the appropriate intervals. the appropriate intervals. Test
Categories 2-4:14 and 28 days. 1 mL or 1 g of the product at 1 mL or 1 g of the product if
0, 14, and 28 days. Record any validated. Record any marked
marked changes in the product, changes in the product, color,
color, odor, etc. odor, etc.

Journal of GXP Compliance Volume 18 Number 3

Peer Reviewed: Antimicrobial Testing
PROCEDURE Test by the pour plate method Titration of the viable cell Remove a suitable sample Remove a suitable sample
and incubate bacteria and yeast counts is based on the pour from each container, typically from each container, typically
for 3-5 days. Incubate mold plate method from “Microbial 1 ml or 1g, at 0 hour and at 1 ml or 1g, at 0 hour and at
for 3-7 days. Can incorporate Limits Tests”. Can incorpo- appropriate intervals according appropriate intervals according
neutralizers if needed. rate neutralizers to the assay to the type of product. Ensure to the type of product. Ensure
if needed. Need to confirm antimicrobial properties are antimicrobial properties are re-
that neutralizers have no effect removed by dilution, filtration, moved by dilution, filtration, or
on the microorganisms. May or an inactivator. Determine an inactivator. Determine the
perform by filtration if neutral- the number of viable cells by number of viable cells by the
izers do not work to overcome the plate count or membrane plate count or membrane fil-
antimicrobial properties of the filtration method. The proce- tration method. The procedure
product. dure needs to be validated. needs to be validated. Incubate
bacteria and yeast for 3-5 days.
Incubate mold for 3-7 days.

Calculate the log reduction. Express sequential changes in Calculate the log reduction. Calculate the log reduction.
the viable counts as percentag-
es the count at the start of the
test is 100.

NA Category 2 products are the NA Methods must be validated to

same as Category 1 but special ensure both uniform disper-
procedures and considerations sion of the microbes and the
are required for both uniform titration of viable cell counts.
dispersion of the test micro-
organisms in the product and
titration of viable cell counts.

NA For semisolid ointment bases, NA For semisolid ointment bases,

heat the sample to 45ºC to heat the sample to 45C to 50C
50ºC until it becomes oily, until it becomes oily, disperse
disperse cells suspension by cells suspension by stirring
stirring with a rod or spatula. with a rod or spatula. Surfac-
Surfactants may be added to tants may be added to disperse
disperse cells evenly. Neutral- cells evenly. Neutralizers may
izers may also be added. also be added.

CRITERIA No increase is defined as When the results below are ob- When the results below are ob- No increase is defined as
not more than a 0.5 log unit tained, the product examined tained, the product examined not more than a 0.5 log unit
higher than the previous value is considered to be effectively is considered to be effectively higher than the previous value
measured. preserved. preserved. In justified cases measured.
where the A criteria cannot
be obtained, the B criteria can
be used.

Category 1: NLT 1.0 log from Category 1A] Bacteria: After Parenteral and Ophthalmic Injections, other parenterals
initial at 7 days, NLT 3.0 log re- 14 days 0.1% of inoculum Preparations: Bacteria A) 6 including emulsions, otic
duction from the initial count count or less/ After 28 days the hours= 2 log/ 24 hours=3log/ product, sterile nasal products,
at 14 days, and no increase same level or less after 14 days. 28 day=no recover Fungi A) 7 and ophthalmic products made
from the 14 days at 28 days Yeasts/ molds: Same or less day=2 log/ 28 day=no increase/ with aqueous bases or vehicles.
for bacteria. Yeast and molds- than inoculum count after 14 Bacteria B) 24 hours=1 log/
No increase from the initial days/ Same or less than inocu- 7 day= 3 log/ 28 day= No in- Recommended Results may be
calculated count at 7, 14, and lum count after 28 days. crease Fungi B) 14 day= 1 log/ modified if needed to incorpo-
28 days. 28 day= no increase. rate option B of the EP.

Bacteria @ 6 hours= 2 log

24 hours=3log
7 days= NLT 3.0 log from
initial count.
14 days= NLT 3.0 log from
initial count.
28 day=no recover

Fungi @ 7 day=2 log

14 days= no increase
28 day=no increase

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CRITERIA Category 2: Bacteria- NLT Category 1B] Bacteria: After Topical Preparations: Bacteria Topically used products,
2.0 log reduction from the 14 days 1% of inoculum count A) 2 day= 2 log/ 7 day=3log/ nonsterile nasal products, and
initial count at 14 days and no or less/ After 28 days the same 28 day=no recover Fungi emulsions, including those
increase from the 14 day count level or less after 14 days. A)14 day=2 log/ 28 day= no applied to mucous membranes.
at 28 days. Yeast and mold- No Yeasts/ molds: Same or less increase/ Bacteria B) 14 day= 3
increase from the initial calcu- than inoculum count after 14 log/ 28 day= no increase Fungi Recommended Results may be
lated count at 14 and 28 days. days/ Same or less than inocu- B) 14 day= 1 log/ 28 day= no modified if needed to incorpo-
lum count after 28 days. increase. rate option B of the EP.
Category 2] Bacteria: Same
or less than inoculum count Topical Preparations:
at 14 days/ Same or less than
inoculum count at 28 days. Bacteria @2 day= 2 log
Yeast/Molds: Same or less than 7 day=3log
inoculum at 14 days/ same or 28 day=no recover
less inoculum count at 28 days.
Fungi @ 14 day=2 log
28 day= no increase

Category 3: Bacteria- NLT 1.0 Category 1C] Bacteria: After Oral preparations: Bacteria Oral products other than ant-
log reduction from the initial 14 days 10% of inoculum 14 days= 3 log/ 28 days=no acids, made with aqueous bases
count at 14 days and no in- count or less/ After 28 days the increase. Fungi: 14 days= 1 or vehicles.
crease from the 14 days count same level or less after 14 days. log/ 28 days= no increase
at 28 days. Yeast and molds- no Yeasts/ molds: Same or less Recommended Results may be
increase from the initial calcu- than inoculum count after 14 modified if needed to incorpo-
lated count at 14 and 28 days. days/ Same or less than inocu- rate option B of the EP.
lum count after 28 days.
Bacteria @14 day= 3 log
28 days=no increase

Fungi @14 days= 1 log

28 days= no increase

Category 4: Bacteria, NA NA Antacids made with an aque-

Yeast, and Molds- No ous base.
increase from the initial Bacteria, Yeast, and Molds- No
calculated count at 14 increase from the initial calcu-
and 28 days. lated count at 14 and 28 days.

One can visualize from Table 2 that it is ubility properties, pH, water activity, and months, then quarterly, then yearly) until
possible to create one method that can be antimicrobial properties will provide valu- the end of life of the product. These deci-
utilized to satisfy all three compendial re- able information during the method devel- sions will often be made by the project team
quirements. This is dependent upon the opment trials. including regulatory and QA, and not sole-
application. For example, if a company ly by the testing laboratory.
only wants to market in the United States, Knowing the composition of product
they do not need to comply with all of the including preservatives in the formulation The risk-based approach necessitates
requirements listed for the USP, EP, and/or can aide in making risk-based decisions on consideration of the following:
JP. test methods and testing frequency. For • What is the concentration of preserva-
example, it is a known requirement to test tive (if any) in the finished product?
It is essential to learn the product and the product during the development phase • Will the preservative be effective in fin-
the excipients so that testing decisions can and at the final stability time point to show ished product
be made. Effective strategies to learn about that the product was effective throughout • Is the preservative toxic at product
the product include collaborating with the the life cycle. Depending on the product concentration?
research and development (R&D) team, specifics (regulatory commitments, punc- • Is the manufacturing process going to
joining the product project team, and lis- ture studies, freeze/thaw studies, acceler- reduce the microbial load?
tening to the chemist working on the proj- ated stability studies, etc.), a company may • What is the water activity?
ect. Discovering how the product is used, decide to test for antimicrobial effective- • Is the water activity level going to pre-
the target patient population, maximum ness during development and at higher fre- vent proliferation of microorganisms if
dose, delivery routes, pharmacology, sol- quency (for example, monthly for several they are present or introduced?

Journal of GXP Compliance Volume 18 Number 3

Peer Reviewed: Antimicrobial Testing
• Do any non-preservative excipients adding neutralizers to the media or utiliz- • An antibiotic that targets gram neg-
have natural anti-microbial properties? ing the membrane filtration method. Vali- ative bacteria needs to be tested. The
• Is the product aqueous-based? dating AET is similar to that of the Micro- product is readily soluble in water.
bial Limits Test Validation. • The product is diluted in phosphate
The assembled information in then used buffer pH 7.2 and the membrane filtra-
to wrote the stability specifications. Docu- The compendial chapters outline useful tion method is chosen.
mented specifications will facilitate method information for the development process as • Because the product targets gram neg-
development and validation processes to be well: ative bacteria, varying dilutions and
streamlined. Regulatory, QA, and stability • If the product contains antimicrobial rinsing agents are utilized with gram
personnel should be consulted when pre- activity, this should be neutralized (9). negative bacteria for the method de-
paring these specifications. • If inactivators are used, their efficacy velopment trials.
and their absence of toxicity for micro- • After an acceptable method has been
ANTIMICROBIAL EFFECTIVE- organisms must be demonstrated (9). identified, the full compendial panel
NESS TEST PERFORMANCE • Common neutralizing agents and of microorganisms are performed to
There are two commonly used enumer- methods include the addition of poly- assure that all of the required micro-
ation methods listed in the compendial sorbate, the addition of lecithin, and/ organisms can be recovered accurately
chapters. Methods include the membrane or dilution methods (9). utilizing the employed method.
filtration method and the plate count meth-
od. Both of these methods are described in Depending on the nature of the product, This is the right time to learn the prod-
USP <61>, which is harmonized with the choose the appropriate method discovered uct or excipient:
EP and JP. Whichever method is utilized in the method development trials. Most • How does the material react to varying
depends on factors such as the nature of common methods include the membrane scenarios?
the product, filtration, and other consider- filtration method and the plate count meth- • How does the material dissolve?
ations. od. • How easily/accurately are inoculated
microorganisms recovered?
The compendial methods are validated In the membrane filtration method, fil- • Does the pH need to be adjusted?
by compendial scientists. However, suit- tration must be performed with filters that • Which method (membrane filtration
ability of the methods to recover microor- have a pore size not greater than 0.45μm or pour plate) is the right method?
ganisms if they are present must be estab- (9). The type of filter material is chosen in • Which neutralizers are needed, if any?
lished. This is really method verification, such a way that the bacteria-retaining ef-
although the term validation is commonly ficiency is not affected by the components By this point, one should have identified
used. of the sample (9). Common filter mate- a method, a diluent, a dilution factor (if
rials include cellulose, nylon, and Polyvi- needed), a sample preparation, any neutral-
Method development trials are the ap- nylidene fluoride (PVDF). izers, and how to adjust the pH. It is also
propriate time to experiment with new recommended to test the water activity of
products. These trials will inform a micro- Regardless of whether one uses the pour the material. Knowing the water activity is
biologist of the reaction to diluents, heat, plate method or the membrane filtration not a requirement for the validation, but it
filtration, sonication, shaking, etc. Method method, it is imperative to prove the suit- is a useful tool. Water activity is discussed
development trials are typically recorded in ability of the test to recover microorgan- in USP <1112> Application of Water Activ-
laboratory notebooks and are supportive to isms. To do this appropriately, the media ity Determination to Nonsterile Pharma-
future validation exercises. utilized must be properly growth promoted ceutical Products. Microorganisms may
as described in the compendial chapter such still be present at levels of <0.6 aw, but they
Method development is initiated by as USP <61>. Proper positive and negative will not proliferate. The test aids in the de-
utilizing information about the product controls along with titer plates must also cisions relating to the following:
that has been gathered from project teams, be utilized throughout the process. Fresh • Optimizing product formulations to
scientists, or chemists. Utilizing known microorganism dilutions must be used (no improve antimicrobial effectiveness of
product information should decrease the more than five passages from the original preservative systems
amount of testing and other effort during master seed lot) (1). Microorganisms may • Reducing the degradation of active
the method development trials. For ex- be purchased from vendors in a ready to pharmaceutical ingredients within
ample, if a product is not soluble in water, use format or prepared as described in the product formulation susceptible to
addition of surfactant such as polysorbate compendial chapters. chemical hydrolysis
or using isopropyl myristate to dissolve the • Reducing the susceptibility of formu-
drug may be tried. If the pH is acidic, it To save time and money during the ini- lations (especially liquids, ointments,
may need to be adjusted to a neutral range tial method development trials, use a subset lotions, and creams) to microbial con-
(pH 6-8) to recover microorganisms. If a of microorganisms to test the product reac- tamination
product has antimicrobial activities, try tions. For example:

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Crystal Booth

• Providing a tool for the rationale for • Once data are collected and a reason- no exception. In fact, EP 5.1.3 describes
reducing the frequency of microbial able method has been identified, the that the designated microorganisms can
limit testing and screening for objec- method and data should be reviewed. be substituted with environmental isolates.
tionable microorganisms for prod- The method will be repeated during It is recommended that E. coli ATCC 8739
uct release and stability testing using validation. If must be able to be suc- be used for oral preparations and Zygo-
methods contained in the general cessfully executed without failures. If saccharaomyces rouxii NCY 381 for oral
chapters <61> and <62> a method is validated in the United preparations containing a high concentra-
• Reducing water activity to greatly as- States, it should be repeatable in Japan. tion of sugar (4). knowing the product is
sist in the prevention of microbial pro- extremely important. If utilizing environ-
liferation.(11) ANTIMICROBIAL EFFECTIVE- mental isolates, do not add spore-forming
• The information could potentially be NESS TEST METHOD bacteria into non-aqueous based products.
utilized in determining the AET fre- VALIDATION The vegetative cells may die, but the spores
quency during stability studies. The validation (also known as method could possibly thrive and cause an out of
verification) exercises should be performed specification investigation. Instead, choose
After the method development data under method validation protocols that are the environmental isolates carefully and
has been evaluated, choose the method in controlled like other cGMP documents. use scientific rationale as to why or why not
which the compendial microorganisms can Typical analytical validations consider the
be recovered by at least 50% of the positive certain isolates will be utilized in the study.
following: Clear documentation of the decision mak-
controls. • Accuracy ing process is recommended.
• Precision
Other points to consider:
• Repeatability Method validation is typically per-
• Is the method one that any analyst can
• Intermediate Precision formed on three lots of material to demon-
easily perform without easily contami-
nating the sample? • Specificity strate the robustness of the method. In
• Are the recovery counts at least 50% of • Detection Limit order to perform the assay, prepare the
the positive controls? • Quantitation Limit sample to be tested according to the pro-
• Growth should not be inhibited (re- • Linearity tocol. Where pouring TSA and SDA are
duction by a factor greater than two) • Range utilized, the media must be tempered to ap-
(9). proximately 45°C (warm to the touch, but
• Does the pH need to be adjusted for There is an ongoing industry expecta- not hot) prior to use.
the validation? If so, the pH will need tion that environmental isolates be incor-  
to be adjusted for testing. porated into Microbiology assays. AET is Table 3 lists the incubation schemes uti-
lized in AET.
Table 3: AET Incubation Scheme

MEDIA MICROORGANISMS INCUBATION TEMPERATURE TIME FRAME

Tryptic Soy Agar (TSA) Escherichia coli ATCC 8739
Bacteria Pseudomonas aeruginosa ATCC 9027 30-35°C 3-5 days
Staphylococcus aureus ATCC 6538
Sabouraud Dextrose Agar (SDA)
Yeast Candida albicans ATCC 10231 3-5 days
Sabouraud Dextrose Agar (SDA)
Mold Aspergillus brasiliensis ATCC 16404 20-25°C 3-7 days

For simplicity, all SDA plates may be read at 3-5 days and still be in compliance. If an analyst chooses to incubate the mold plates longer than the yeast plates, all SDA
controls must be incubated for the longer time interval.

Good microbiology practices must be em- AET Method Examples • Use SDA for Molds and incubate at
ployed. All equipment, Petri dishes, pipette To prepare the inoculum, grow each of 20-25°C for one week or until good
tips, diluents, etc. must be sterile. Good the test microorganisms on the appropri- sporulation occurs but not exceeding
aseptic technique is required in order to ate media. Either plates or slants may be 10 days.
obtain accurate results. used. The viable microorganisms will not
be more than five passages removed from Wash the growth off of the plates or slants
Lyophilized microorganism preparations the original ATCC culture. with approximately 4.0 mL of sterile saline
purchased from vendors may be utilized for • Use TSA for bacteria and incubate at 0.85% (use sterile saline 0.85% with 0.05%
AET. Single-strain challenges are utilized 30-35°C for 18-24 hours. Tween 80 for Aspergillus brasiliensis) and
in this assay. Other strains or species that • Use SDA for Yeasts and incubate at 20- collect in a sterile test tube. Dilute the mi-
may represent likely contaminants to the 25°C for 48 hours. croorganisms to a concentration of ~1x108
product may also be added to the protocols.

Journal of GXP Compliance Volume 18 Number 3

Peer Reviewed: Antimicrobial Testing
CFU/mL. This may be accomplished by and adding it into another tube of 9.9 Method development or special testing
using the turbidimetric technique, as the mL of diluent. Vortex the solution must be documented in method develop-
dilutions need to be fresh. For best results well. ment notebook following good documen-
with the turbidimetric technique, it is rec- • Perform a 1:10 dilution by adding 1.0 tation practices. This documentation may
ommended to use a mid to upper range 0.5 mL of the previous dilution into 9.0 be requested during a regulatory audit. Af-
McFarland for bacteria and a mid to upper mL of diluent. Vortex the solution ter a method has been developed, a valida-
range 3.0 McFarland for yeast. It is recom- well. tion protocol should be written.
mended to prepare a very dark solution for
molds, >3.0 McFarland (e.g. ~5-10% light Plate the appropriate dilutions in duplicate The method evaluation portion of the
transmittance). in order to bracket the theoretical count- validation will prove the method allows for
able colonies (typically found at 10-5, 10-6, recovery of microorganisms if they are via-
Suspensions should be used immediately. and 10-7). Pour tempered TSA over bac-
ble. This only needs to be performed for the
The actual concentration of the suspensions teria and tempered SDA over yeast and
0 hour interval during the first three anal-
may be determined by preparing serial di- molds. Allow the plates to solidify at room
yses of AET. At Validation Completion, it
lutions and pour plates. When calculating temperature and then incubate accordingly.
the N0 (or titer) of the saline suspension, is recommended to write a Validation Re-
the dilution diluent for bacterial and yeast To determine the N0 of the saline suspen- port to summarize the validation and then
suspensions are 0.85% Sterile Saline. The sion, average the two plates, which contain a specific method for each product should
dilution diluent for A. brasiliensis is 0.85% between 30 and 300 colonies and back cal- be written.
Sterile Saline with 0.05% Tween 80. The di- culate. Adjust the original saline suspen-
luents may change depending on the com- sion with the appropriate dilution diluent Where the product container can be en-
pendia being utilized. to yield approximately 1x108 CFU/mL. See tered aseptically (such as with a needle and
Figure 1. syringe through a rubber stopper), conduct
Make serial dilutions of the saline suspen- the test in the original product contain-
sion and adjust when needed. Record all of the information obtained. If ers. If the product container is such that it
• Pipette 0.1 mL of the suspension into Quanti-cults are utilized, the passage num- cannot be entered aseptically, or sufficient
a tube of 9.9 mL of diluent. Vortex the ber is typically listed on the microorganism volume is not present, transfer an appro-
solution well. This is a 1:100 dilution. package label. priate amount (depending on the method
• Perform one more 1:100 dilution by development) of the product to a suitable
taking 0.1 mL of the previous dilution container.
Figure 1: Example Calculation: 0.2 mL inoculum into 40 mL of Product
If the product is a large volume, an
appropriate quantity (depending on the
method development) may be transferred
to a smaller container or the inoculum may
be added proportionally (example: 0.1 mL
per 20-mL product volume). There should
be a container with the minimum amount
of product for each microorganism to be
inoculated and a least one negative control
if the volume is sufficient for the remainder
of the assay.
N0= Countable Average Plate Counts is multiplied by the dilution factors.
0.1 mL has a dilution factor of 10.
Inoculate each container with 0.1 mL
0.01 mL has a dilution factor of 100.
(or more as determined by the volume of
1:10 dilution has a dilution factor of 10.
product) of each of the challenge microor-
1:100 dilution has a dilution factor of 100.
ganism inoculums prepared and label the
containers accordingly. There should be
In the above example 51 is the countable average plate count.
at least one un-inoculated product control
No = 51 X 10 X 10 X 100 X 100
container. The volume of inoculum and
= 5.1 X 107 CFU/mL
product in the sterile containers may be ad-
justed as long as the volume of the suspen-
CFU/mL of Product= N0 / (divided by) Dilution Factor / (divided by) amount of sion inoculum used is between 0.5% and
Product in mL X (multiplied by) 2 (to double the 0.1 mL results). 1% of the volume of product. Example: A
= 5.1 X 107 CFU/mL / 10 / 40 X 2 container with 40 mL of product would be
= 2.6 X 105 CFU/mL (Correct inoculum achieved) inoculated with 0.2 mL of inoculum.

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Crystal Booth
Mix the solution well to ensure a ho- Perform negative controls on un-inoc- Include one negative control TSA plate and
mogenous distribution of the microor- ulated samples by testing product without one negative control SDA plate during the
ganisms. Determine the number of viable the addition of microorganisms. Plate incubation process.
microorganisms in each inoculum suspen- un-inoculated aliquots into duplicate petri
sion by referencing the results from the N0 dishes. Cover one duplicate set with tem- Incubate all samples according to Table 3.
(or titer). Then, calculate the initial con- pered TSA and one duplicate set with SDA.
centration of microorganisms per mL of Also, perform negative controls on any me- Count the colonies on each product plate
product under test. The target inoculum dia and/or diluents used in test. Incubate and compare to the colonies of the positive
should yield a suitable concentration be- one TSA and one SDA plate as media con- control plates. Calculate the percent recov-
tween 1x105 and 1x106 CFU/mL of prod- trols. Incubate all negative controls accord- ery using the formula: CFU of product test
uct. Except for antacids where the target ing to the temperatures and times indicated plates/CFU of positive control plates X 100%.
inoculum is 1 X 103 to 1 X 104 CFU/mL of for the media type in Table 3. The percent recovery should be at least 50%.
Product according to the USP. Record the  
results and record any changes observed in Count the colonies and record the re- Testing Intervals
the appearance of the product throughout sults. Calculate the log reduction and re- The testing intervals outlined in the USP and
the test. cord the results. EP was merged into Table 4 so that all testing
points are accounted for each product type.
Pour Plate Method Example. Samples Example calculation:
should be taken at 0 hours (immediately Log Reduction Table 4: Testing Time Intervals
after inoculation) and at time intervals out-
lined in Table 4. At each time interval after = Log of Initial Calculated CFU/mL – Log TESTING INTER-
the sample aliquot has been taken, the re- of Product Challenge Results CFU/mL PRODUCT PRODUCT VALS COMPLY-
CATEGORY DESCRIPTION ING WITH THE
maining product in the container is placed = Log of 1 X 105 CFU/mL – Log of 1.0 X USP AND EP
back into the 20-25°C incubator (protecting 102 CFU/mL (or 100 CFU/mL) Injection, Other Bacteria: 0 hour,
it from light) until the next time interval. Parenterals Includ-
=5–2=3 6 hour, 24 hour, 7
ing Emulsions, Otic days, 14 days, and
Mix the product. Dilute, if necessary, Products, Sterile 28 days
so that 1.0 mL (or 1.0 g) will be expect- Membrane Filtration Method Evaluation. 1 Nasal Products,
ed to yield between 30 and 300 colonies For each microorganism and product and Ophthalmic Fungi: 0 hour, 7
control, prepare test tubes containing the Products Made days, 14 days, and
(if growth is not inhibited). Pipet 1.0 mL with aqueous bases
(or weigh 1.0 g) of the product (or dilutions appropriate amount of un-inoculated 28 days
or vehicles.
if needed) into each of two sterile petri product (or un-inoculated product dilu- Topically used
tion) and label accordingly. Pipette 0.1 mL Bacteria: 0 hour,
dishes. products made 2 days, 7 days, 14
(<100 CFU) of each microorganism into with aqueous days, and 28 days
To each petri dish containing bacteria, separate ~100 mL aliquots of the diluent bases or vehi-
utilized in the method development, such cles, nonsterile
promptly add approximately 15 to 20 mL 2
nasal products,
of tempered TSA. Likewise, add tempered as Fluid A. and emulsions,
Fungi: 0 hour, 7
days, 14 days, and
SDA to plates containing yeast and molds. including those 28 days
Note that larger Petri dishes and larger vol- Following the filtration procedure devel- applied to mucous
umes of media with neutralizers may be oped during method development. Filter membranes.

used if needed as determined by the meth- an un-inoculated product aliquot through Oral Products Bacteria: 0 hour, 14
a sterile 0.45µm filter. Rinse the filter with other than antacids, days, and 28 days
od development process. Cover the Petri 3
made with aqueous
dishes and swirl to distribute the microor- the total amount diluent volume. The last Fungi: 0 hour, 14
bases or vehicles. days, and 28 days
ganisms evenly. Allow the media to solidify ~100 mL rinse will be the inoculated dilu-
at room temperature. Invert the dishes and ent preparation containing <100 CFU of Bacteria: 0 hour, 14
Anatacids made days, and 28 days
incubate according to Table 3. microorganisms as described previously. 4
with an aqueous
base. Fungi: 0 hour, 14
Prepare positive control plates to use as days, and 28 days
Perform positive controls by diluting
the inoculum solution (without product) a reference by filtering the same volume Regardless of the testing method employed,
so that 1.0 mL will be expected to yield be- of diluent without the product. The last the acceptance criteria should confirm either
tween 30 and 300 colonies. Inoculate 1.0 rinse will be another diluent preparation to the most stringent compendial require-
mL of the diluted inoculum solution into inoculated with <100 CFU of the micro- ments if a hybrid method is used or to the tar-
each of two sterile Petri dishes. Incubate organism to be tested. Filter two negative geted compendia. In the examples described
the plates according to Table 3. The posi- controls of the product without microor- in this article, the acceptance criteria conform
tive controls will serve as “growth promo- ganisms. Also filter two un-inoculated to the USP and EP. The worst case results
tion” of the media as well as to prove that samples of ~100 mL of diluent to serve as were chosen between both Pharmacopeia and
the microorganisms are initially viable. negative diluent controls. Place one filter are presented in Table 5. All specifications are
on TSA and one filter on SDA. given in terms of log reduction.

Journal of GXP Compliance Volume 18 Number 3

Peer Reviewed: Antimicrobial Testing
Table 5: USP and EP Specifications

PRODUCT CATEGORY PRODUCT DESCRIPTION TESTING SPECIFICATIONS COMPLYING WITH THE USP AND EP
0hour 6hour 24hour 7days 14days 28days
Injection, Other Parenterals Including Emulsions, Bacteria
1 0 2 3 3 3 No Recovery
Otic Products, Sterile Nasal Products, and Ophthal-
mic Products Made with aqueous bases or vehicles. Fungi 0hour 7days 14days 28days
0 2 No Increase No Increase
0hour 2days 7days 14days 28days
Topically used products made with aqueous bases or Bacteria 0 2 3 3 No Increase
2
vehicles, nonsterile nasal products, and emulsions,
including those applied to mucous membranes. 0hour 14days 28days
Fungi
0 2 No Increase
0hour 14days 28days
Bacteria
3 Oral Products other than antacids, made with aque- 0 3 No Increase
ous bases or vehicles. 0hour 14days 28days
Fungi
0 1 No Increase
0hour 14days 28days
Bacteria
0 3 No Increase
4* Anatacids made with an aqueous base.
0hour 14days 28days
Fungi
0 1 No Increase
*In the case of Category 4 products, the EP does not distinguish between Oral Products and Antacids. Therefore, the EP specification is given as it is more stringent.

In Table 5, the EP gives allowances for category 1 and 2 specifications. It breaks down the specifications into parts A and B. Part A
is given in Table 5 because the specification is more stringent. In justified cases where part A cannot be attained (e.g. increased risk of
adverse reaction), part B must be satisfied. Part B is given in Table 6.

Table 6: Part B Specifications to Comply with USP and EP in the Event of Justified Cases Where Part A Is Unattainable

PRODUCT CATEGORY PRODUCT DESCRIPTION TESTING SPECIFICATIONS COMPLYING WITH THE USP AND EP
0hour 6hour 24hour 7days 14days 28days
Injection, Other Parenterals Including Emulsions, Bacteria
1 0 0 1 3 3 No Increase
Otic Products, Sterile Nasal Products, and Ophthal-
mic Products Made with aqueous bases or vehicles. Fungi 0hour 7days 14days 28days
0 0 1 No Increase
0hour 2days 7days 14days 28days
Topically used products made with aqueous bases or Bacteria 0 0 0 3 No Increase
2
vehicles, nonsterile nasal products, and emulsions,
including those applied to mucous membranes. 0hour 14days 28days
Fungi
0 1 No Increase

Any out of specifications (OOS), should It is difficult sometimes to have three lots of Acceptance Criteria
be investigated according to company pro- clincal product in order to perform a com- Validation acceptance criteria are es-
cedures. These investigations should in- plete validation. Small amounts of product sential when determining if a method was
clude the identification of microorganisms are often made for use in clinics. Formu- properly validated. When verifying the
(if applicable). lations change frequently depending on suitability of the membrane filtration or
field studies and developments discovered the Plate-Count Method, “a mean count of
Commercial Products and Clinical throughout the early phases of a project. It any of the test organisms not differing by a
Products is acceptable to perform method validation factor greater than 2 from the value of the
The question of how to treat clinical on one lot for clinical products. The com- control in the absence of product must be
products verses commercial products is a pany will likely be redeveloping and revali- obtained” (9). In other words, the percent
debated tropic. Most companies agree that dating with each formulation change of the recovery between the inoculated product
the industry practice is to validate a mini- product. It may take years for a product to dilutions and the positive controls must be
mum of three lots for commercial product reach commercial launch. However, when at least 50%. Some companies use an in-
method validations. product moves to commercial manufactur- ternal 70% recovery criterion that is accept-
Clinical products are treated differently. ing, a three-lot validation must be initiated. able because it is more stringent.

www.ivtnetwork.com
Crystal Booth

The inoculated plates prepared during When developing methods, be creative. 7. Moser, Cheryl L. and Brian K. Meyer “Compar-
the method evaluation portion of the val- Learn as much as possible from develop- ison of Compendial Antimicrobial Effectiveness
Tests: A Review” AAPS PharmSciTech Vol. 12,
idation must be acceptable in order for the ment scientists, chemists, research, and No. 1, March 2011.
validation to be valid. The titer plates must project teams to aide in the method de- 8. Sutton, S. “Antimicrobial Efficacy Test”, PMF
demonstrate not more than 100 cfu was uti- velopment trials. Utilize the information Newsletter, March 2010.
lized to achieve the positive results (9). This to target the method development trials 9. USP Chapter <61> “Microbiological Examina-
is necessary so that the method employed in order to save time and minimize costs. tion of Nonsterile Products: Microbial Enumer-
ation Tests”
can demonstrate that the method is capable Once a suitable method has been identified, 10. USP Chapter <1111> “Microbiological Exam-
of recovering low levels of microorganisms perform a trial as one would do during the ination of Nonsterile Products: Acceptance
if they are present. validation with all of the microorganisms Criteria for Pharmaceutical Preparations and
to demonstrate the method will work prop- Substances for Pharmaceutical Use”
After validations are complete, stabili- erly. Perform these trials in a notebook and 11. USP <1112> “Application of Water Activity De-
termination To Nonsterile Pharmaceutical Prod-
ty testing may begin. Reports are usually keep thorough and detailed notes about the ucts”
written to approve the studies and methods trials. Choose the best suitable method for 12. ICH Harmonised Tripartite Guideline: Valida-
(or standard operating procedures [SOPs]) the application. Do not underestimate the tion of Analytical Procedures: Text and Method-
are written to lockdown the way the tests value of knowing the water activity. ology Q2(R1)
are routinely performed. 13. ICH Harmonised Tripartite Guideline: Bracket-
ing and Matrixing Designs for Stability Testing
During validation, write a method vali- of New Drug Substances and Products Q1D
To determine the frequency of test- dation protocol utilizing the method devel-
ing, one can utilize a risk-based approach. oped in the trial runs. Be certain that the
This is going to be a product-by-product method is performed the same way each About the Author
risk assessment. ICH QA6 provides some time and that it is repeatable. For clinical
guidance on determining the appropriate products, a one lot validation is sufficient Cystal Booth is the Microbiology Labo-
testing intervals. “In general, it is advisable due to various clinical constraints. For ratory Manager at Covidien in Raleigh,
to test the drug product unless its compo- commercial products, a minimum of a NC. She earned her Bachleor’s Degree in
nents are tested before manufacture and the three lot validation should be performed. Biology from Old Dominion University
manufacturing process is known, through and her Master’s of Microbiology Degree
validation studies, not to carry a significant Create a clean validation package for from North Carolina State University. She
risk of microbial contamination or prolif- regulatory audits. During routine testing, has 14 years of experience in Pharmaceu-
eration” (6) document the method so that it is per- tical Microbiology. Crystal has developed
formed the same way every time. Brack- and performed numerous method val-
Stability testing is performed under var- eting or matrixing can be utilized in de- idations. Some of the methods include
ious temperatures and humidity set-points. creasing the volume of stability testing. Microbial Limits Testing, Bacterial Endo-
AET is typically performed for stability Risk-based approaches should be utilized toxins Testing, Particulate Testing, Steril-
testing during product development, at ini- in determining the testing frequencies. ity Testing, Pharmaceutical Water System
tial, yearly, and then end of life in multidose Clear documentation and justifications Validations, Environmental Monitoring
containers. When designing the program, should be maintained so that decisions can Programs, Surface Recovery Validations,
take into account the product formulations, be clearly understood in the future. Disinfectant Efficacy Studies, Minimum
strengths, and packaging configurations. It Inhitory Concentration Testing, Anitmi-
may be appropriate to leverage bracketing crobial Effectivesness Testing, Hold Time
REFERENCES Studies, and various Equipment Valida-
or matrixing stability testing designs as
allowed under ICH Q1D. Bracketing or
1. USP Chapter <51> “Antimicrobial Effectiveness tions (Autoclaves, Isolators, Vitek, Biolog,
Testing” EM equipment, Conductivity Meters, pH
matrixing can greatly reduce the test sam- 2. Sutton, S. “The Antimicrobial Efficacy Test, GMP
ples needed for the stability program. With and Investigations” Microbiology Network July/
Meters, Pipettes, PTS Unit, Endoscan,
acceptable scientific justification, it may be August 2013. http://microbiologynetwork.com/ TOC equipment, Incubators, etc.). Crys-
possible to propose AET during develop- content/APR_2013_Sept_Antimicrobial-Effec- tal has worked in both R&D and Quality
ment and end of life testing only. Another
tiveness-Test-GMP-Investigations.pdf Control Laboratories, including a Start-
3. Sutton, Scott and David Porter “Development up Company. She has developed and
option is to fill the product in a single use of the Antimicrobial Effectiveness Test as USP
dosage form in order to propose bypassing validated methods for Antibiotics, Otics,
Chapter <51>” PDA Journal of Pharmaceutical
the test for antimicrobial effectiveness. Science and Technology, Vol. 56, No. 6, Novem- Topical Creams, Topical Ointments, Oral
ber/December 2002 Solid Dose Products, Oral Liquid Dose
CONCLUSIONS 4. EP 5.1.3. Efficacy of Antimicrobial Preservation Products, Veternary Products, Human
5. JP XVI (19) Preservatives-Effectiveness Tests Parenterals, Asepticallly Filled Products,
Learn about the material before begin- (PET)
ning. Set appropriate specifications so that and Terminally Sterilized Products. She
6. ICH Guidance Q6A- Specifications: Test Pro-
the method can be appropriately targeted. cedures and Acceptance Criteria for New Drug has experience working with global mar-
For international compliance, use the most Substances and New Drug Products: Chemical kets and regulatory bodies.
Substances
stringent international method, or a mix-
ture of the methods.

Journal of GXP Compliance Volume 18 Number 3